Membrane arrays and methods of manufacture

ABSTRACT

The invention relates to G protein-coupled receptor (GPCR) microarrays on porous substrates for structural or functional analyses of GPCRs, and methods of preparing porous substrate surfaces for receiving membranes that comprise GPCRs. In one embodiment, a GPCR microarray of the invention comprises a membrane adhered to an upper surface of a porous substrate, the membrane spanning across a plurality of pores on the porous substrate to form a plurality of cavities having sufficient geometry to permit entry of assay reagents into each cavity, thereby allowing access of assay reagents to both sides of GPCR in the membrane.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to G protein-coupled receptor (GPCR)microarrays on porous substrates for structural or functional analysesof GPCRs, and methods of preparing porous substrate surfaces forreceiving membranes that comprise GPCRs.

2. Background of Related Art

GPCRs are the single most important class of drug targets—approximately50% of current drug targets are membrane bound. Despite the large numberof GPGR targets and a wide variety of technologies for screening againstGPCRs, no methods were available for screening against multiple GPCRssimultaneously. The GPCR microarray technology has been investigated(Fang et al., “G Protein-coupled Receptor Microarrays for Drug Delivery”Drug Discovery Today, Vol. 8, No. 16, August 2003, pp. 755-761; Bieri etal., “Micropatterned Immobiliztion of a G Protein-coupled Receptor andDirect Detection of G Protein Activation,” Nature Biotechnology, Vol 17,November 1999, pp. 1105-1108; Pierce et al., “Seven-TransmembraneReceptors” Molecular Cellular Biology, Vol. 3, September 2002, pp639-650) and their use has been demonstrated for the multiplexedscreening of compounds, see for example U.S. Patent ApplicationPublication Nos. 2002/0019015 and 2002/0094544, the entire disclosuresof which are hereby incorporated by reference.

The arrays were obtained on flat “2D” glass slides coated withγ-aminopropylsilane (GAPS) and other materials includingepoxypropylsilane. Most assay development has focused on “bindingassays” that provide information about how much of a compound is boundto a receptor at a particular concentration; based on this information,the affinity of the compound for the receptor can be obtained.

A large fraction of GPCR screening assays—so-called “functionalassays”—are based on determining whether the GPCR gets activated as aresult of compound binding. The information can be used to classifycompounds as agonists, partial agonists, antagonists or inverseagonists. Moreover, functional assays are essential for investigating“orphan” GPCRs, some of which may turn out to be key drug targets.Orphan GPCRs are those without known ligands, which preclude the use ofcompetition assays employing known labeled ligands. Functional assayscan be both cell-based and biochemical in nature; cell-based assays arecurrently the method of choice for functional assays. Cell based assaysinclude reporter gene assays, β-arrestin and GPCR-GFP translocationassays (i.e., receptor internalization and endosome formation). Methodsfor monitoring the activation of GPCRs by non-cell based assays aremostly limited to monitoring GTP-GDP exchange at the GPCR associated Gαprotein using labeled GTP analogues (e.g., ³⁵S-GTPγS or Eu-GTP). Thesefunctional assays are “homogenous” assays, that is, the receptor and theGTP analogue mixed with or without a compound of interest are insolution over the duration of the assay; these assays are then subjectto filtration using a filter microplate so that the labeled GTP can beremoved by filtration, and only the bound GTP analog molecules can bequantified and the effect of compound on the binding of GTP analog canbe examined which can be used to classify the action of compound on thereceptors (i.e., non-binder, or antagonist, or agonist, etc).

Limited success has been encountered with the use of fluorescent-dyelabeled GTP-YS for functional assays on 2D GAPS surfaces, althoughfunctional assays employing radioactively labeled ³⁵S-GTPγS have beensuccessfully carried out on these surfaces. However, the relatively poorreproducibility of these functional assays limits their applications ofGPCR microarrays for compound screening. Moreover, the use ofnon-radioactive labels is preferred because of safety issues.Europium-labeled GTP (Eu-GTP) (Perkin Elmer Life Science, Boston, Mass.)has been developed as an alternative to ³⁵S-labeled-GTP, and has beensuccessfully demonstrated their use in functional assays carried out insolution in combination with filter-plates. Realization of Eu-GTPbinding assays for GPCR microarrays on porous substrates would greatlybenefit their applications for compound screening.

With regard to the production and use of GPCR microarrays, the G proteincoupled receptor (GPCR) microarrays are unique in that they requireimmobilization of both the protein targets and the lipid membrane inwhich they are embedded (Fang et al., “Membrane Protein Microarrays,” J.American Chemical Society, Vol. 124, 2002, pp. 2394-2395; Fang et al.,“G-Protein Coupled Receptor Microarrays,” Chembiochem., Vol. 3, 2002,pp, 987-991). Moreover, the confined proteins should be in theircorrectly folded conformations. Different types of surfaces have beenproposed that meet these requirements (Hennestal et al., “Pore SpanningLipid Bilayers Visuallized by Scanning Force Microscopy,” J. AmericanChemical Society, Vol. 122, 2000, pp. 8085-8086; Cremer et al.,“Formation and Spreading of Lipid Bilayers on Planar Glass Supports,” J.Physical Chemistry B, Vol. 103, 1999, pp. 2554-2559; Theato et al.“Formation of Lipid Bilayer on a New Amphiphilic Polymer Support,”Langmuir, Vol. 16, 2000, pp. 1801-1805; Majewski et al., “StructuralStudies of Polymer-Cushiond Lipid Bilayers,” J. Biophysical Journal,Vol. 75, 1998, pp. 2363-2367.

Conventional methods for fabricating solid supported membranes exploitgold-thiol, capping of OH-groups by silanes, and electrostaticinteractions. The resulting membranes exhibit limited long-termstability due to the lipid loss into the solution when remained inaqueous solutions (Fang and Yang, “The Growth of Bilayer Defects and theInduction of interdigitated Domains in the Lipid-Loss Process ofSupported Phospholipid Bilayer,” Biochim. Biophys. Acta, Vol. 1324,1997, pp. 309-319), but their close proximity to the solid surface(typically 0.2-2 nm) limits lateral lipid mobility. Since amembrane-surface separation of at least 1 to 5 nm (preferably at least 2to 10 nm) is usually required to preserve the biological functions ofthe membrane proteins associated with the membranes, several approacheshave been employed to extend the membrane surface distance, such as theuse of lipids with long hydrophilic spacers, the inclusion of polymercushions between substrate and membrane, and the use of patterns withvaried thiol-components that increase lateral mobility and free volume.

A functional GPCR assay is possible if both GPCR terminals areaccessible and bioactive. Suspended membranes have been developed on thebasis of membranes spanning the pores of porous alumina substrates(Hennestal et al., supra). When the membrane spans the pores there areno issues with steric congestion on either side of the receptor.

A method has been proposed which makes use of “contact printing” todeposit a binding chemistry, such as a moderately positively chargedcoating, only onto the top surface of a porous substrate. The contactprinting includes impregnating a flat polymer stamp with a solutioncontaining the active molecules and brings it in conformal contact withthe porous substrate. This effectively transfers the active moleculesonly onto the top surface of the substrate.

Further, it is also known to perform functional assays for G-ProteinCoupled Receptors (GPCRs) in commercially available 96 well plates usinga time-resolved fluorometric assay based on GDP-GTP-Eu-labeled exchangeon GPCR. The activation of receptors by agonists is made in solutioninside wells. The activation signal is detected on the porous bottom ofwells where the activated receptor of the GPCR is retained afterfiltration (see, for example, the DELFIA GTP-binding kit from PerkinElmer Inc.).

G-protein coupled receptor (GPCR) microarrays are unique in that theyallow immobilizing both the protein targets and the lipid membrane inwhich they are embedded before activation. One advantage of thistechnique is to use small amounts of expensive receptors and to studyseveral receptors simultaneously in the same well. However, the confinedprotein should be in their correctly folded conformations. Differenttypes of surfaces have been proposed that meet these requirements asdiscussed above.

Therefore, it can be realized that effective GPCR microarrays for use infunctional assays, e.g., employing particular GTP analogues, are needed.Additionally, a simple method of selecting the appropriate poroussubstrate for receiving a membrane, enhancing the immobilization of themembrane on the porous support, as well as a simple method of fixing amembrane on the porous support, are needed.

SUMMARY OF THE INVENTION

The present invention overcomes the problems of forming reliable GPCRmicroarrays described above by providing GPCR arrays on poroussubstrates for use in functional assays. Membrane arrays comprisingother transmembrane proteins can be similarly prepared.

In one aspect, the present invention provides a membrane array whichincludes (1) a porous substrate comprising a plurality of pores; and (2)a plurality of membranes adhered to the porous substrate. Thesemembranes comprise transmembrane proteins that are accessible to assayagents from both sides of the membrane. Any type of membrane can be usedfor the present invention, such as biological membrane (e.g., plasmamembrane, nuclear membrane, or cell organelle membrane), reconstitutedmembrane (e.g., liposome, or other unilaminar or multilaminaramphiphilic molecule complexes), or polymer complexes (e.g., hydrogel).These membranes can be either covalently or non-covalently attached tothe porous substrate. The transmembrane proteins can be any protein ofinterest, such as G protein-coupled receptors (SPCRs), ion channels,receptor kinases, or transporters. In many examples, each transmembraneprotein includes a ligand-binding domain located on one side of themembrane and an effector-binding domain located on the other side of themembrane.

In one embodiment, each membrane on a membrane array of the presentinvention comprises a lipid bilayer, and each membrane at leastpartially spans across one or more pores in the porous substrate. Thesurface properties of the porous substrate and these pores satisfy thefollowing set of relations:

γ_(lw)+γ_(ls)+2γ_(pw)−2γ_(lp)−γ_(sw)<0 and

γ_(ls)<γ_(lw)+γ_(sw)

wherein,

-   -   γ_(lw)=surface tension of the lipid-assay medium interface;    -   γ_(ls)=surface tension of the lipid-substrate interface;    -   γ_(pw)=surface tension of the pore-assay medium interface;    -   γ_(lp)=surface tension of the lipid-pore interface;    -   γ_(sw)=surface tension of the substrate-assay medium interface.

In another embodiment, each membrane on a membrane array of the presentinvention at least partially spans over two or more pores in the poroussubstrate to form a plurality of cavities. Each cavity has geometry topermit access of assay reagents into the cavity.

In another aspect of the invention, a process is set forth for creatinga bi-functional porous substrate in which the process involves threesteps. In the first step, the porous layer is coated by a non-bindingchemistry. Then, the top surface of the porous layer is exposed toultraviolet radiation in the presence of ozone to oxidize or remove theorganic coating on the top surface of the porous substrate. Thereafter,another binding chemistry is deposited on the bare top surface to createa bifunctional porous substrate.

Finally, an embodiment of the invention also includes a technique of“contact printing” a GPCR-embedded membrane on a porous substrate inwhich, preferably, a binding chemistry procedure has been previouslyperformed. The binding chemistry procedure can, for example, providingthe upper surface of the porous substrate with a moderately positivelycharged coating onto the upper surface of a porous substrate. However,other binding chemistry procedures well known in the prior art canequally be employed. The contact printing of the invention includes thesteps of impregnating a flat polymer stamp with a solution containingactive molecules, and bringing the stamp into conformal contact with theupper surface of the porous substrate to effectively transfer the activemolecules onto the top surface of the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be further understood by reference to the drawings,wherein:

FIGS. 1A and 1B, show an idealized representation of a GPCR microarrayon: (A) 2-dimensional substrate and (B) porous substrate;

FIG. 2 shows an example of a GPCR array functional assay on GAPS coatedporous slide in which human neurotensin receptor subtype 1 (NTR1),opioid receptor mu subtype (Opioid Mu) and motilin receptor (Motilin)were printed in array format on porous slide;

FIGS. 3A and 3B illustrate a pore-spanning configuration (top) in whichboth GPCR terminals are accessible and bioactive; while, in thepore-coating configuration (bottom), the inner C-terminal is squeezedbetween the membrane and substrate and, therefore, is not available forfunctional assay;

FIG. 4 is a schematic diagram of a pore-spanning membrane desirable forfunctional assays;

FIG. 5 is a schematic diagram of a pore-coating membrane, which is notdesirable for functional assays;

FIG. 6 illustrates a porous substrate untreated with a non-bindingcompound which has been dipped in Fibrinogen solution and stained withcolloidal gold solution;

FIG. 7 illustrates a porous substrate treated over its entire surfacewith a binding compound which has been dipped in Fibrinogen solution andstained with colloidal gold solution;

FIG. 8 illustrates a porous substrate, having a GAP coating only on theentire surface, after gold staining;

FIG. 9 illustrates a porous substrate of the invention having received anon-binding compound and exposure to ultraviolet radiation in presenceof ozone and GAPS coating only on the top surface, after gold staining.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves fabrication of GPCR microarrays on poroussubstrates in order to perform “functional” GPCR assays. It is notedthat the ability to carry out functional assays indicates thefeasibility of use in binding assays, such as that shown in patentapplication US 2002/0094544A1, on these porous substrates. Therefore,the instant invention is not limited to providing only functional assaysof the GPCR-type, but can be directed to any assay in which exposure ofboth ends of a protein, across a membrane, is preferred for accurateresults. Examples of these proteins include, but are not limited to, ionchannels, transporters, kinase receptors, or other transmembraneproteins.

Although conventional protein microarrays provide direct informationregarding the binding and selectivity of putative drugs, they fall shortin their ability to predict biological function. The biophysicalrequirements to study ligand agonism or antagonism using microarrays arechallenging—a molecule has to bind to an immobilized protein in themicrospot, the protein has to then undergo a conformational change thatleads to the binding of a second molecule at a different binding site.Yet, two-site tandem binding is the paradigm for the activation ofcell-surface receptors.

For binding assays, it is preferred that the ligand binding domain ofthe GPCR (located on the extracellular N-terminus and/or theextracellular binding sites formed by the membrane spanning loops of thereceptor) be exposed to the assay solution (containing the labeledligand and potential drug compounds). For GPCR containing membranepreparations immobilized on GAPS (or other amine containing surfaces),we assume that 50% of the immobilized receptors have their ligandbinding domains facing the solution with the intracellular G-proteinbinding domain face down on the GAPS surface. GPCRs may also beimmobilized in an oriented manner. For example, a GPCR with its ligandbinding domain exposed to the solution (“facing up”) could be obtainedusing a GPCR biotinylated (or histidine tagged) at its C-terminusprinted on a streptavidin (or N-chelate) coated surface; whereas a GPCRwith its G-protein side facing up could be obtained throughimmobilization via its glycosylated N-terminus on wheat-germagglutinin-coated surfaces, The latter orientation may be useful ifmonitoring GPCR-G protein interactions is desired.

For functional assays, access to both sides of the receptor ispreferred. In the resting state of the receptor, GDP is bound to theG_(α) subunit of the heterotrimeric G protein (Gp) associated with theGPCR. Upon activation due to ligand binding, GDP dissociates from theactivated complex and is replaced by GTP (or its analogue). Therefore,equilibrium is achieved between the GPCR-Gp complex, the ligand (L),GDP, and the GTP analogue (GTPS♦), as shown in Equations 1 and 2.

GPCR−Gp+L=L−GPCR*−Gp−GDP  (Eq 1)

L−GPCR*−Gp−GDP+GTPS♦=L−GPCR+Gp−GTPS♦  (Eq 2)

Partitioning of GTPS♦ into the interstitial spaces of a supportedmembrane can be hindered (FIG. 1A), especially if the label attached tothe GTP is bulky. Porous surfaces that lead to the formation ofsupported membranes spanning the pores (FIG. 1B) of the substrate canalleviate this steric congestion and enable access to both sides of theimmobilized receptor. That is, the agonist is able to access the ligandbinding domain of the GPCR and GTPS♦ should be able to simultaneouslybind to the G_(a) subunit of Gp (FIG. 1B).

There are additional reasons why porous surfaces are important forfunctional assays. The increased binding capacity of the surface maylead to the immobilization of a larger number of receptors relative toflat surfaces. Although the binding affinity of a ligand for a GPCRdepends on whether it is complexed to a G protein, the overall bindingsignal is an average signal obtained from complexed and uncomplexedforms (the use of nanaomolar concentrations of ligands biases the assayto the complexed form) of the receptor. Functional assays work for thosereceptors complexed with right heterotrimeric G-protein. Anotheradvantage in using porous substrates is the potential for opticalenhancements due to scattering by the porous microstructure or due tothe strong enhancement of the incident light within the nanopores underresonant conditions (Liu, Y. and Blair, “Fluorescence enhancement froman array of subwavelength metal apertures”, Optics Letters, 2003, Vol,28, 507). Since the net signal upon functional activation is relativelylow, substrates that offer higher sensitivity (assuming that the assayis not limited by non-specific binding) would facilitate the monitoringof GPCR activation.

As discussed further below, the invention describes a process of formingporous surfaces coated with GAPS (or any surface presenting polymerscontaining amino groups can also be used), the fabrication of GPCRmicroarrays on these substrates, and functional assays on these arraysthat monitor the agonist mediated binding of europium labeled GTP(Eu-GTP) to the microspots form at the pores of the porous substrate.

The process of fabricating and employing the GPCR functional assay ofthe invention includes three major steps:

1) Array printing—GPCR membranes are printed on GAPS coated porousslides using contact (solid pin or quill pin-based) or non-contact(injet, bubble injet or nanoliter liquid dispenser) printingtechnologies. Preferably, the printed slides are then subject topost-printing treatments. These treatments include about one-hourincubation under about 75% humidity in a humidity chamber and followedby drying for about two hours under vacuum at room temperature beforeassay;

2) Assays performing—the printed GPCR arrays are then incubated withassay buffers containing GTP analogue probes in the presence or absenceof receptor agonist for certain time (e.g., 45 minutes), and at the endof incubation period the assay solutions are removed, the slides arerinsed with GTP wash buffer and dried with nitrogen;

3) Imaging—when Eu-GTP is used as a probe, the slides are then directlyscanned using a time-resolved fluorescence imaging system; and, ifbiotin-GTPγS is used as a probe, an extra incubation step is oftenperformed before imaging. For example, the array is then furtherincubated with Gold particles labeled streptavadin in order to detectbounded biotin-GTPγS, resonance light scattering imaging system is thenused.

FIG. 2 illustrate an example of a GPCR array functional assay on GAPScoated porous slide of the invention in which NTR1, Opioid Mu andMotilin receptors were printed in array format on porous slide. Aftertwo hours of vacuum drying, the arrays were incubated for 1 hour withEuGTP assay buffer in the absence or presence of receptor agonists(neurotensin for NTR1, motilin for Motilin receptor, and dynorphin A forOpioid Mu; each agonist is at 1 μM). The EuGTP assay buffer contained 10nM Eu-GTP, 50 mM HEPES buffer, pH 7.4, 3 μM GDP, 10 mM MgCl₂, 100 mMNaCl, and 0.1% protein blocker. At the end of the incubation, the assaybuffers were removed. Afterwards, slides were washed, dried and imagedwith in-house developed time-resolved fluorescence imaging system. Thedegree of activation (%) is plotted for a control and the variousreceptors. The results suggest that the presence of three agonists (eachfor its cognate receptor) increase the binding of EuGTP to the GPCRmicrospots in the arrays, indicating that the GPCRs in the arrays areactivated by the agonists.

The use of porous substrates for functional assays using GPCRmicroarrays of the invention will remove the stringent restrictions onthe size of GTP analogues and any other molecules that can recognize orbind specifically to activated receptors (e.g., beta-arrestin) oractivated G proteins (e.g., G protein-binding peptides), and therebyprovide high quality, high sensitivity functional assays in a highlymultiplexed manner.

Additionally, enhanced detection schemes such as those using resonancelight scattering (e.g. using GTPγS-biotin, followed by streptavidincoated gold nanospheres) or signal amplification schemes (e.g. usingGTPγS-biotin, followed by streptavidin-HRP for amplification) can alsobe used with the functional assays of the invention. Another embodimentof the invention involves parallel performing binding and functionalassays by applying an assay solution containing a GTP analogue as wellas labeled ligand(s) with known functional properties (agonist versusantagonist) in the absence or presence of a compound of interest. TheGTP analogue gives signals in one channel (e.g., time resolvefluorescence signal of Eu-GTP, or radioactivity signal), whereas thelabeled ligands give signals in other channels (e.g., FITC, Cy3 orothers). Each labeled ligand binds specifically and selectively to itscognate receptor(s) in the microarrays.

Another embodiment of the invention involves a process of selecting theappropriate porous substrate for use with a particular membrane. In theregard, the invention further includes defining the surface chemistriesof the substrate and pore to favor membrane spanning conformations forthe GPCR-embedded membranes over an arbitrarily sized pore in the poroussubstrate. These surface chemistries are based on an analysis of thecompetition between interfacial energies involving the membrane lipids,substrate, pore, and aqueous environment.

While the focus of this invention is on GPCR array functional assays, itmust be realized that the invention is also useful for theimmobilization of any bio-layer or membrane (e.g., biological membrane,reconstituted membrane, or polymer such as a hydrogel) when theapplication requires steric access to both sides of the bio-layer ormembrane.

As mentioned above, functional GPCR assays yield far greater informationabout the effectiveness of drug candidates than binding assays alone,and functional GPCR assays are greatly facilitated by obtaining accessto both sides of the GPCR containing membrane. Porous substrates havebeen identified as candidate surfaces on which a functional GPCR assayscan be designed. The present invention enables membrane spanning overarbitrarily sized pores, since the surface designs are to be based onthe surface chemistry of interaction. That is, the present invention isindependent of the specific coating materials that may be used, and,hence, provides an extremely broad and powerful scope of applications.

As can be seen in FIGS. 3A and 38, it is beneficial for theGPCR-receptor to be accessible to the assay medium at both surfaces ofthe membrane in order to provide reliable binding and results. Forsubstrates with conformal lipid membrane, FIG. 3B, the exposure to theassay medium of the C-terminus end of the GPCR-receptor adjacent thesubstrate surface is hindered by the close association, e.g., physicalcontact or squeezing, of the membrane and porous substrate.

Without limiting the present invention to any particular theory, ananalysis of the difference in free energy between the pore-spanningconfiguration is illustrated in FIG. 4, and the pore-coatingconfiguration is illustrated in FIG. 5, which is given by:

${{\Delta \; G} = {{\pi \; {d^{2}\left( {\gamma_{lw} + \gamma_{ls} + {2\; \gamma_{pw}} - {2\; \gamma_{lp}} - \gamma_{sw}} \right)}} - {4\; \pi \; {K_{c}\left( {1 + \frac{\pi \; d}{2\; t}} \right)}}}},$

where:

d=radius of the pore;

γ_(lw)=surface tension of the lipid-water interface;

γ_(ls)=surface tension of the lipid-substrate interface;

γ_(pw)=surface tension of the pore-water interface;

γ_(lp)=surface tension of the lipid-pore interface;

γ_(sw)=surface tension of the substrate-water interface;

K_(c)=the bending modulus of the membrane; and

t=the thickness of the membrane,

A negative value of ΔG indicates that the pore-spanning configuration isfavorable; hence, a negative value of ΔG is desirable for functionalassays. If we assume the worst-case scenario for pore-spanning, i.e. aperfectly fluid membrane, we may set K_(c)=0. To allow pore-spanning inthis worst-case scenario, we preferably have for a hemispherical pore,

πd ²(γ_(lw)+γ_(ls)+2γ_(pw)−2γ_(lp)−γ_(sw))<0.

Therefore, the ability of a membrane to span a pore can be a function ofthe surface tension of the substrate and of the pore, and not a functionof the pore geometry:

γ_(lw)+γ_(ls)+2γ_(pw)−2γ_(lp)−γ_(sw)<0.

If this condition is satisfied, then the pore-spanning membraneconfiguration is favorable relative to the pore-coating configuration.In many instances, the condition γ_(ls)<γ_(lw)+γ_(sw) is also satisfiedto ensure binding of the membrane to the substrate.

Therefore, many embodiments of the present invention are directed to theselection of a porous surface in which the surface chemistries are suchthat both the conditions γ_(lw)+γ_(ls)+2γ_(pw)−2γ_(lp)−γ_(sw)<0 andγ_(ls)<γ_(lw)+γ_(sw) are satisfied. If the geometry of the pore-shapewere generalized, then the first condition above can be generalized towrite:γ_(lw)+(S_(p)/S_(d)−1)(γ_(ls)−γ_(sw))−S_(p)/S_(d)(γ_(lp)−γ_(pw))<0,where S_(p) refers to the pore surface area and S_(d) refers to thepore-spanning membrane area of the pore. For instance, for the idealhemispherical pore, S_(p)=2πd²,S_(d)=πd², we obtain the earliercondition.

Therefore, as long as these two inequality conditions are satisfied, thepores may be of any size and geometry suitable for functional assays.

Other hypotheses may also explain why porous substrates enablefunctional assays for GPCR or other membrane proteins. Biologicalmembranes are unstable on bare (unmodified) flat glass substrates. See,e.g., Cremer et al., supra. Moreover, the use of bare glass substratesdo not offset the membrane by a distance (e.g., about 2 nm or less) fromthe surface that enables the folding of extramembrane domains. However,bare (unmodified) porous glass supports offer mechanical stability andenable specific binding of ligands to GPCR arrays. See, e.g., U.S.patent application entitled “Porous Substrate Plates and the UseThereof” (by Ye Fang et al., Attorney Docket No. SP04-026). The presentinvention indicates that these types of supports also enable functionalassays. Specifically, the present invention demonstrates that GPCRmicroarrays on organic polymeric porous supports enable GPCR functionalassays. Many theories are available to explain why porous substrates areexcellent candidates for functional assays. In addition to theabove-described theories, one hypothesis is that porous substratesenable multilayer deposition of membranes in structures such that thetortuosity enforced by the substrate satisfies the requirement foraccess to both sides of the membrane.

Once the proper selection of porous substrate has been made, it is oftennecessary to provide the surface of the substrate with the ability tosecurely receive membranes. One method of providing a bifunctionalporous of the invention is to apply a non-binding coating on the entireporous substrate. Then the non-binding coating is oxidized (or burned)at the top surface using an ultraviolet (UV) lamp. The UV lamp, e.g.,mercury lamp, emits UV radiation at two different wave lengths, e.g.,185 and 254 nm, to produce ozone and oxidize organic contaminants.Thereafter, a second coating material including silane is grafted on theburned top surface to provide the binding or immobilization sites forGPCR.

This results in a structure in which the top surface of the poroussubstrate binds to the membrane; while the membrane and GPCR do not bindor adhere to the inner walls of pores which contain the non-bindingcoating material.

It is known that slightly positively charged surfaces are usuallysuitable to bind membranes to surfaces. Such surfaces may be obtained bygrafting organic groups with amine functions to the substrate surface.If the substrate has a porous layer made from a glass frit, anaminosilane is suitable to modify the glass surface.

The hydrophilic, non-binding properties of the inner pore walls may beobtained if, for example, the untreated glass substrate is hydrophilicor coated with a non-binding coating. Several alternatives can be used.For example, the inner pore walls may be coated with a silane terminatedpolyethylene glycol (PEG-silane) or with a hydrolyzed epoxy silane.

In a specific method of the invention, the porous substrate is entirelycoated by dipping the substrate in a solution containing an organicpolymer with a large amount of hydroxyl functions, which provide the nonbinding properties desired, e.g., a hydrophilic coating is formed. Thispolymer is prepared by polymerization and hydrolysis, in very acidicconditions (pH=0), of, for example, an epoxysilane(3-Glycidoxypropyltrimethoxysilane, called GLYMO). After 1 minute ofdipping, the chemical condensation of the polymer is accelerated by athermal treatment of 1 hour at 70° C. Then, the excess of polymer notbound onto the substrate is eliminated by rinsing with a water flow for30 seconds and drying with nitrogen,

The non-binding properties of this coating can be checked by dipping thesubstrate in a fibrinogen aqueous solution (0.1% in PBS). Fibrinogensare proteins, strongly sticking to glass surface and glass frit porouslayers. The adsorbed proteins can then be revealed by a colloidal goldstaining, as shown in FIGS. 6 and 7, wherein FIG. 6 illustrates thebinding of fibrinogen on a clean porous substrate and FIG. 7 illustratesvirtually no binding for porous substrate provided with a non-bindingcompound.

When the non-binding compound coated porous substrate is subjected to anoxidation treatment in the presence of ozone (formed from oxygen exposedto UV at 185 nm which is associated with UV at 254 nm), burning of thenon-binding organic compound from the upper surface of the treatedsubstrate results. In many cases, the thickness of the layer to beburned is only a few molecular layers. For example, one hour of exposureof a coated sample under this lamp results in a “cleaned” upper surface,free of organics and presenting reactive SiOH sites.

This surface can be used for direct binding of GPCR or for furtherfunctionalization of the top surface, e.g., with silane.

After the burning of the non-binding coating at the top surface of theporous substrate, the 3-aminopropyltriethoxysilane (called GAPS), whichprovides amine functions for further GPCR-containing membraneimmobilization, is applied using a vapor phase (CVD) deposition process.The presence of GAPS silane grafted onto the top surface can be revealedwith a colloidal Gold staining.

FIGS. 8 and 9 illustrate the results of performing the above invention.That is, the clean porous substrate only coated with GAPS (FIG. 8)presents an intense gold staining on the porous areas; while thebi-functionalized porous substrate of the invention, with a GAPS coatingonly at the top surface, presents a lighter gold stain. This result ispresumed to occur due to a thinner layer of porous substrate beingcoated with GAPS silane, i.e., the thinner layer corresponding to thethickness of the non-binding coating burned with the UV lamp producingozone.

Still embodiment of the invention includes providing the poroussubstrate upper surface with binding capability, e.g., GAPS silane, inorder to immobilize membranes thereon.

This result may be obtained when the top surface of the porous substratebinds to the membrane and when the membrane and the GPCR do not bind oradhere to the inner walls of pores.

As mentioned above, slightly positively charged surfaces are usuallysuitable to binds membranes. Such surfaces may be obtained by graftingorganic groups with amine functions to the substrate surface. If thesubstrate has a porous layer made from a glass frit, an aminosilane issuitable to modify the glass surface. Amine functionality may be alsoobtained on a glass surface by first grafting an epoxysilane, and thenderivatizing the epoxy groups with a diamine or polyamine to form anaminated surface.

The hydrophilic, non-binding properties of the inner pore walls may beobtained if the untreated glass surface is hydrophilic or coated with anon-binding coating, as in FIG. 9. To provide a membrane receptivesurface, the application of, for example, functional silanes, on solidsurfaces by a dry-printing technique has been described. The processincludes impregnating a silicone rubber stamping pad, which bears a maskpattern in relief, with octadecyltrichlorosilane, and then in pressingthe pad against a flat surface of silicon or of oxides of metals such asTi and Al. A reaction takes place between the silane and the coatedsurface such that the silane molecules are bound to the surface via oneof their ends. This surface is then subjected to chemical etchingintended to attack the parts which are not protected by the silane mask.

The instant invention provides for a simpler and more effective processof forming the membrane receptive surface on porous substrates whichprovides a coating of molecular thickness on a three-dimensional, poroussubstrate by dry or contact printing of a compound having an affinityfor the substrate. The transfer element whose impregnated surface isflat and uniformly impregnated is placed in contact with a substratecontaining relatively high exposed parts (upper porous surface) andrelatively low recessed parts (pores), so as to selectively apply thecompound/coating onto the high exposed parts of the substrate, leavingthe low recessed parts essentially free of compound.

The dry printing process may typically be carried out by performing thefollowing steps:

(a) providing a transfer element with a flat surface made of a rubberymaterial capable of undergoing swelling under the effect of an organicsolvent;

(b) preparing a solution, in an organic solvent, of a compound having anaffinity for the substrate, e.g., capable of bonding to or associatingwith the substrate surface by any mechanism such as chemical bonding,attraction of opposite electrical charges, or hydrogen bonding;

(c) applying the solution to the clean flat surface of the transferelement, and allowing the transfer element to absorb the solutioncompletely, Evaporation of the solvent may also take place;

(d) pressing the surface of the transfer element treated with thesolution against a clean porous substrate and leaving in contact untilthe molecules of the compound are bonded to the surface of the substratethereby forming a coating of molecular thickness bonded to therelatively high exposed parts (upper surface) of the porous surface ofthe substrate; and

(e) separating the transfer element from the substrate.

When the coated compound is an organosilane, it may be one selected fromtwo different types of groups. The first type includes thoseorganosilanes which have a functional group that reacts with groupspresent on the substrate surface. The second type of functional groupsis not reactive with a hydroxyl group, in contrast with the first type.

One class of compounds which can be used in the practice of theinvention is those silanes of the general formula R_(n)—Si—X_(4-n),where R is a functional group which is not reactive with a hydroxylgroup; X is a group which is reactive, and/or which is hydrolyzable intoa group which is reactive, with a hydroxyl group, and n=1, 2 or 3. Forexample, R may be an epoxy group or a radical containing the epoxy groupor an amino group or a radical containing an amino group. X may be, forexample, a chlorine atom or an alkoxy group, such as methoxy and ethoxy.

Specific examples of silanes which can be used in the invention include,but are not limited to,

-   (3-glycidoxypropyl)trimethoxysilane,-   (3-glycidoxypropyl)methyidimethoxysilane,-   (3-glycidoxypropyl)methyldiethoxysilane,-   (3-glycidoxypropyl)dimethylethoxysilane,-   3-aminopropyltriethoxysilane,-   3-aminopropyltrimethoxysilane,-   4-aminobutyltriethoxysilane,-   N-(2-aminoethyl)-3-aminopropylmethyidimethoxysilane,-   N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,-   N-(6-aminohexyl)aminorpropyltrimethoxysilane,-   3-aminopropyidimethyethoxysilane,-   3-aminopropylmethyldiethoxysilane.

The compound may also be any organic molecules whose groups react with asilane coated substrate. For example, in an alternative to theabove-described method for providing a surface for immobilizing themembrane, the porous substrate may be first entirely coated with anepoxysilane, and then the top surface of the porous layer mayselectively react with a polyamine deposited by dry printing. Thistechnique is essentially the same as for the selective deposition of anyorganosilane. However, in the alternative, the organic compound reactswith the epoxy functionality of the silane coated porous layer. As aresult, the top surface of the substrate has amine groups and theinternal part of the porous layer stays coated by the epoxy silane. In alast step, the epoxy functions can be hydrolyzed as to form ahydrophilic, uncharged, and non-binding surface inside the porosity.

The transfer element can be made of any solid or solid-like materialcapable of undergoing swelling under the action of an organic solvent.For example, a rubber such as a silicone, polyisoprene, polybutadiene orpolychloroprene rubber; butadiene-styrene, butadiene-acrylonitrile,ethylene-propylene or ethylene-vinyl acetate elastomeric copolymers;butyl rubber, and polysulfide rubber. However, a silicone rubber ispreferred.

The organic solvent may be any solvent capable of dissolving thecompound to be deposited and of exerting a swelling effect on thematerial of the transfer element. An example of those types of solventswould be liquid alkanes such as hexane, heptane, octane, decane andhexadecane; halogenated alkanes such as chloroform, aromatic compoundssuch as benzene or toluene; petroleum fractions such as white spirit,diesel oil, gasoline and other solvents such as tetrahydrofuran andN-methylpyrrolidone. Indeed, most organic solvents may be suitable forthe invention and a simple routine test will make it possible to checkthe usefulness of a given solvent. It suffices to impregnate thetransfer element with small amounts of compound and, for this, verydilute compound solutions are sufficient.

The porous substrate can be any material whose surface bears hydroxylgroups. An example of those type of substrate would be glass, silica,metals, or polymers whose surface has been modified to create hydroxylgroups thereon, for example by a chemical oxidizing treatment or with aplasma, or alternatively coated with a layer of glass, silica or metalby techniques such as sputtering, chemical deposition in the vaporphase, or sol gel. In many cases, the pore size of the substrate isgreater than 0.05 μm.

The compound solution may be applied to the transfer element, or polymerstamping pad, in various ways, for example by rubbing an absorbent papersoaked with the solution onto the transfer element, by rubbing a porousmaterial, such as a sponge, soaked with the solution onto the transferelement, by applying the solution using a doctor blade or an air blade,a sprayer or a coating roller.

This process of the invention may be used to impart adhesion to therelatively high exposed parts (upper surface) of the surface of a poroussubstrate, containing hydroxyl groups, to a membrane. The inner walls ofthe porous substrate have to be hydrophilic and uncharged to developnon-binding properties, such as by the process of described for theporous substrate of FIG. 9 above. Clean bare porous glass substrates maybe suitable; however, if not, a non-binding chemistry can be depositedor developed inside the porous structure, as described above. Severaldifferent options for performing the process of this invention areillustrated by the following examples.

EXAMPLE 1

A porous bi-functional substrate may be obtained from a glass slidecoated with a porous layer. The porous layer can be made from a glassfrit having the appropriate particle size to form pores having adiameter greater than 0.05 μm, typically of the order of 1 μm aftersintering. The top surface of the porous structure is coated by dryprinting with aminopropylsilane. Thus the top surface of the substratebecomes slightly positively charged to retain the membrane. The innerpart of the porous structure stays as a hydrophilic, hydroxyl richsurface, which may not allows binding of the GPCR protein and membraneinside the pores.

EXAMPLE 2

As an alternative to example 1, after having dry printed the top surfaceof the porous substrate with an aminosilane, the inner part of theporous structure can be coated with a silane terminated PEG. Thisnon-binding coating will be grafted onto uncoated glass part, e.g., inthe inner part of the porous structure.

EXAMPLE 3

A slide coated with a porous layer with a pore size of the order of 1 μmis completely coated with an epoxy silane, such asglycidoxypropyltrimethoxysilane, by liquid or vapor phase deposition.The top surface of the porous structure is selectively derivatized bydry printing a diamine or a polyamine. The diamine may be for exampleethylene diamine, tetraethylene pentamine, or hexamethylene diamine. Theepoxy functions inside the porous structure can be hydrolyzed in acidicconditions to form a hydrophilic, uncharged and non-binding coatinginside the pores.

The foregoing examples of specific compositions, processes, and/orarticles employed in the practice of the present invention are of courseintended to be illustrative rather than limiting, and it will beapparent that numerous variations and modifications these specificembodiments may be practices within the scope of the appended claims.

1-10. (canceled)
 11. A method of making a membrane microarray for usewith an assay medium in which the microarray includes a porous substratehaving a plurality of pores in an upper surface and a membrane at leastpartially spanning over one said pore, the method comprising the stepsof, (a) providing a transfer element having a flat surface thatcomprises a material capable of undergoing swelling under the effect ofan organic solvent; (b) preparing a solution, in the organic solvent, ofa compound having an affinity for the substrate; (c) applying thesolution to the flat surface of the transfer element and permitting thetransfer element to absorb the solution; (d) pressing the surface of thetransfer element treated with the solution against the upper surface ofthe porous substrate until the molecules of the compound are bonded tothe surface of the substrate to form a coating of molecular thicknessbonded to the upper surface of the substrate; and (e) separating thetransfer element from the substrate.
 12. The method according to claim11, wherein the solution is rendered capable of bonding to the uppersurface of the porous substrate by treatment of the porous substrate bya technique selected from the group consisting of coating the substrateto enhance chemical bonding, applying an opposite electrical charge tothe substrate, and chemical treatment of the substrate to effectcovalent bonding to the substrate.
 13. The method according to claim 11,wherein the compound is an organosilane of the formula:R_(n)—Si—X_(4-n), wherein R is a functional group which is not reactivewith a hydroxyl group; X is a group which is reactive and/or which ishydrolyzable into a group that is reactive with a hydroxyl group or anoxide, and n=1, 2 or
 3. 14. The method according to claim 13, wherein Ris an epoxy group or a radical containing the epoxy group, an aminogroup or a radical containing an amino group, and X is a chlorine atomor an alkoxy group.
 15. The method according to claim 11, wherein thetransfer element comprises a solid or solid-like material capable ofundergoing swelling under the action of an organic solvent and isselected from the group consisting of silicone rubber, polyisoprene,polybutadiene rubber, polychloroprene rubber, butadiene-styrene,butadiene-acrylonitrile, ethylene-propylene elastomeric copolymers andethylene-vinyl acetate elastomeric copolymers, butyl rubber andpolysulfide rubber.
 16. The method according to claim 11, wherein theorganic solvent may be any solvent capable of dissolving the compoundand of exerting a swelling effect on the material of the transferelement.
 17. The method according to claim 16, wherein the organicsolvent is selected from the group consisting of liquid alkanes,halogenated alkanes, aromatic compounds, petroleum fractions,tetrahydrofuran and N-methylpyrrolidone.
 18. The method according toclaim 17, wherein the organic solvent is selected from the groupconsisting of hexane, heptane, octane, decane, hexadecane, chloroform,benzene, toluene, white spirit, diesel oil and gasoline.
 19. The methodaccording to claim 17, wherein the porous substrate is a material whoseupper surface bears hydroxyl groups or oxide groups.
 20. The methodaccording to claim 19, wherein the porous substrate comprises a materialselected from the group consisting of glass, silica, metal and polymer.21. A method of making a membrane microarray for use with an assaymedium in which the microarray includes a porous substrate having aplurality of pores in an upper surface and a membrane at least partiallyspanning across one said pore, the method comprising the steps of: (a)coating an upper surface of the porous substrate with a non-bindingcompound; and (b) exposing the upper surface of the porous substrate toultraviolet radiation in the presence of ozone to oxidize the organiccoating on the upper surface of the porous substrate.
 22. The methodaccording to claim 21, further comprising depositing a compound forenhancing the binding chemistry on the upper surface of the poroussubstrate.
 23. The method according to claim 22, wherein the depositedcompound includes an aminosilane.
 24. The method according to claim 21,wherein the non-binding compound includes hydroxyl groups.
 25. Themethod according to claim 21, further comprising the step of providing amembrane adhered to the upper surface of the porous substrate such thatthe membrane at least partially spans over two or more pores of theporous substrate to form a plurality of cavities having sufficientgeometry to permit access within each cavity to the assay medium.
 26. Amethod for identifying a modulator of a membrane protein, comprising:contacting a membrane with a candidate molecule, said membranecomprising said membrane protein and being immobilized on a poroussubstrate, and both sides of said membrane being accessible to assayagents; and detecting a biological function of said membrane protein,wherein a change in said function in the presence of said candidatemolecule as compared to that in the absence of said candidate moleculeis indicative of the capability of said candidate molecule to modulatesaid membrane protein.
 27. The method of claim 26, wherein said membraneprotein is selected from the group consisting of a G protein coupledreceptor, an ion channel, a kinase receptor, and a transporter.
 28. Themethod of claim 26, wherein said membrane is a cellular membrane. 29.The method of claim 26, wherein said membrane protein is a G proteincoupled receptor, and said membrane comprises a G protein or a subunitthereof, and wherein said function is detected by measuring activationof said G protein or subunit by said G protein coupled receptor.
 30. Themethod of claim 29, comprising contacting said membrane with (1) aligand of said G protein coupled receptor and (2) a GTP analogue capableof binding to said G protein or subunit upon activation of said Gprotein or subunit.
 31. The method of claim 30, wherein said candidatemolecule is an agonist of said G protein coupled receptor.
 32. Themethod of claim 30, wherein said candidate molecule is an antagonist ofsaid G protein coupled receptor.
 33. (canceled)